Situ determination of alum filling evenness and sedimentation in pharmaceutical products using water proton nmr

ABSTRACT

A method of using the transverse relaxation rate (R 2 ) of solvent NMR signal to noninvasively assess alum-containing products such as vaccines. This technique can be used for quality control in vaccine manufacturing (e.g., fill-finish step) to determine the evenness of alum filling level as well as extent of alum particle sedimentation in filled and sealed products.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. § 121 and claims priority to U.S. patent application Ser. No. 16/593,145 filed on Oct. 4, 2019, now U.S. Pat. No. 11,585,770, which claims priority to U.S. Provisional Patent Application No. 62/741,142 filed on Oct. 4, 2018 in the name of Yihua (Bruce) Y U et al. and entitled “In Situ Determination of Alum Filling Evenness and Sedimentation in Pharmaceutical Products using Water Proton NMR,” both of which are hereby incorporated by reference in their entirety.

FIELD

The present invention relates to methods for noninvasive quality control of alum-containing products, including vaccines comprising alum adjuvants, using solvent nuclear magnetic resonance (NMR). The methods can be used to assess and the extent of alum particle sedimentation in sealed containers regardless of optical transparency of the alum-containing products.

DESCRIPTION OF THE RELATED ART

Adjuvants play a critical role in the efficacy of vaccines. The most widely used vaccine adjuvants are aluminum salt particles, more commonly referred to as alum. Presently, more than 20 vaccines contain alum adjuvants, including the anthrax, DTaP, DTaP/Hepatitis B/Polio, DTaP/Polio, DTaP/Polio/Hib, Hib, Hepatitis A, Hepatitis A/Hepatitis B, Hepatitis B, HPV, Meningococcus B, Pneumococcus, Tetanus and Diptheria Toxoids Adsorbed, TdaP, and the Diphtheria and Tetanus Toxoids Adsorbed vaccines.

The most commons alum salts include aluminum hydroxide, aluminum phosphate, and aluminum hydroxyphosphate sulfate [Baylor, 2002]. Alum particles are micron-sized with certain size heterogeneity [Lindblad, 2004]. Adjuvant dose in released products is related to vaccine safety and efficacy and adjuvant toxicity is of concern to vaccine safety [Petrovsky, 2015].

Disadvantageously, the alum particles are heavier than water and therefore tend to sediment and phase separate from water [Muthurania, 2015]. Sedimentation during manufacturing may cause the uneven filling of vials, resulting in vaccines with too much or too little alum adjuvant, which results in less than optimal immune responses in immunized subjects (too much may cause harm while too little may be ineffective). Filling levels of alum-adjuvanted vaccines are currently determined using atomic absorption microscopy [Mishra, 2007] or ²⁷Al/³¹P NMR spectroscopy [Khatun, 2018].

Sedimentation during storage is unavoidable and requires shaking to fully resuspend the settled alum particles. Shipping stresses may make re-suspension difficult, with significant vial-to-vial variation [Guo, 2016]. Current qualitative in situ verification of vaccine re-suspension is the shake test [Kartoglu, 2010]. Current quantitative evaluation of vaccine re-suspension includes UV absorption spectroscopy (ex situ) [Guo, 2016], optical scanning analyzer (ex situ) [Muthurania, 2015] and microCT (in situ) [Lewis, 2017]. The ex situ techniques are destructive (as they require taking the drug substance out of its container) and perturbative (as they require dilution, pH adjustment, and other sample preparation steps). The in situ technique, micro-CT, involves ionizing radiation, which may damage the drug substance inside the container. All these techniques require ten minutes or longer for data acquisition as well as highly trained personnel for analysis.

The fundamental problem caused by destructive testing technologies for product quality control (QC) is that only a few units (i.e., vials, pre-filled syringes, etc.) are quantitatively inspected in each lot/batch. This leaves the possibility that defective products escape detection and thus are available to cause harm to patients. Because vaccines are typically administered to healthy people, a few adverse events in a large population cohort may cause panic in the public, leading to a decline in the vaccination rate [Signorelli, 2016; Levi, 2017].

There is a need for a fast and reliable technique which can be used for quality control in vaccine manufacturing, for example, the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in sealed products in the distribution chain, from release to right before injection. Towards that end, the present invention relates to a method of using the transverse relaxation rate (R₂) of the solvent NMR signal, e.g., R₂(¹H₂O), to determine the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in filled and sealed products. Advantageously, the method described herein is easy to use, noninvasive, fast, and highly-sensitive.

SUMMARY

The present invention generally relates to a method of using NMR relaxation rates, specifically the transverse relaxation rate constant R₂ of solvent molecules, e.g., water, to determine the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in alum-containing products such as filled and sealed vaccine products.

In one aspect, a method of determining the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in an alum-containing product is described, said method comprising:

measuring the transverse relaxation rate of solvent R_(2,m) in the alum-containing product; and determining the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in the alum-containing product by comparing the measured R_(2,m) to a reference transverse relaxation rate of solvent R_(2,r) wherein the reference R_(2,r) represents an acceptable range of evenly distributed aluminum particles or suspected aluminum particles, wherein when the measured R_(2,m) is inside the reference R_(2,r) range, the evenness of the alum particle filling levels and/or the alum particle resuspension is acceptable.

Other aspects, features and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Dependence of water proton transverse relaxation rate R₂(¹H₂O) on alum concentration (full suspension) for two alum adjuvants. The insert shows the region of typical alum salt concentration in many vaccine products.

FIG. 2(A). Fully suspended (left) and sedimented (after one week of sedimentation) aqueous suspension of Alhydrogel® in a sealed vial.

FIG. 2(B). Fully suspended (left) and sedimented (after one week of sedimentation) aqueous suspension of Adju-Phos® in a sealed vial.

FIG. 3(A). R₂(¹H₂O) vs. aluminum salt concentration observed for suspended, sedimented and resuspended Alhydrogel®. In each panel, solid and dashed lines represent suspended and sedimented aluminum salt, respectively.

FIG. 3(B). R₂(¹H₂O) vs aluminum salt concentration observed for suspended, sedimented and resuspended Adju-Phos®. In each panel, solid and dashed lines represent suspended and sedimented aluminum salt, respectively.

DETAILED DESCRIPTION, AND PREFERRED EMBODIMENTS THEREOF

The present invention generally relates to a method of using NMR relaxation rates, specifically the transverse relaxation rate constant R₂ of solvent molecules, e.g., water, to determine the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in alum-containing products such as filled and sealed vaccine products.

Advantageously, the method described herein is a reliable and simple method to assess the extent of sedimentation in alum-containing products, e.g., vaccines comprising alum adjuvants, and hence has application as a quality control tool for any field utilizing alum-containing products, e.g., vaccine manufacturing. The method enables the non-destructive assessment of the alum-containing products, formulated as aqueous suspensions, without opening the vial or product container, by measuring the transverse nuclear spin relaxation rate constant, R₂, of a solvent, e.g., water. As will be discussed in detail hereinafter, uneven filling levels of alum particles or the sedimentation of same in the product containers are evidenced by a variation, either an increase or a decrease, of the transverse nuclear spin relaxation rate constant, R₂, of water. The R₂ constant of an alum-containing product, as well an acceptable range of R₂ constants of said alum-containing product (i.e., an acceptable range of evenly distributed aluminum particles or suspected aluminum particles), can be determined by the manufacturer. The acceptable range of R₂ constants can be provided on the package insert, on the vial label, or both. Thereafter, as new batches of alum-containing products are prepared, the R₂ constant of water of each new batch can be measured by the manufacturer to confirm the even filling of alum particles in the product containers before releasing same for sale and purchase. If the measured R₂ constant of the tested alum-containing product is outside of the reference range provided by the manufacturer, meaning that the vaccine dose is either too high or too low due to uneven alum particle filling, the specific vial containing the tested alum-containing product should be rejected. Further, the R₂ constant of water can be measured by the purchaser/user before use to confirm an acceptable resuspension of the alum particles in the alum-containing products pursuant to the manufacturer's guidelines. If the measured R₂ constant of the resuspended alum-containing product is outside of the reference range provided by the manufacturer, meaning that there was unacceptable resuspension, the tested alum-containing product should be rejected. Accordingly, this method redefines the conventional methods of evaluating the uneven filling and/or sedimentation of alum particles in aqueous suspensions comprising alum-containing products, e.g., vaccines, for each product container.

Reference herein to alum-containing products can include vaccines comprising alum adjuvants as well as pharmaceuticals comprising aluminum particles.

Recent breakthrough developments in the instrumentation for nuclear magnetic resonance (NMR) spectroscopy and imaging have opened up opportunities to design novel nondestructive analytical techniques for the nanoparticle industry. The analytical procedures become significantly faster with the application of commercially available computer-controlled NMR autosamplers. Of special importance was the appearance of commercially available, relatively inexpensive benchtop NMR and magnetic resonance imaging (MRI) instruments and relaxometers [Metz, 2008]. Benchtop NMR instruments enable highly accurate measurements of nuclear spin relaxation times T₁ and T₂. Moreover, most of these instruments have a permanent or electronically cooled magnet with the variable bore from 10 mm to 45 mm and even larger which provides a great flexibility in the nonintrusive measurements of vials of various sizes. Other known portable and handheld NMR instruments are known in the art and can be used to practice the methods described herein.

The present inventors have previously shown that the transverse relaxation rate of the water proton NMR signal, R₂(¹H₂O), can be used to monitor solute association in aqueous solutions, such as gadolinium-chelate clustering inside hydrogels [Weerasekare, 2011], stiffness of hydrogels [Feng, 2011; U.S. Pat. No. 9,348,008], protein aggregation and surfactant micellization [Feng, 2015], insulin aggregation [Taraban, 2015], protein concentration [Yu, 2017], monoclonal antibody aggregation [Taraban, 2017a], nanoparticle clustering [Taraban, 2017b], and amide hydrolysis [Briggs, 2018]. Water proton NMR (wNMR) monitors water, which acts as a reporter for analytes dissolved in it. As a reporter, water has two tremendous advantages. First, its concentration far surpasses that of any analyte dissolved in it, by 10³-10⁶ fold in most cases. This makes the ¹H₂O signal easily detectable by benchtop and other portable NMR instruments. Further, the solute association can be detected through the solvent NMR signal. Second, water is “endogenous” to all biomanufacturing processes and all biologic products, including vaccines. This sets it apart from “exogenous” reporters such as fluorescent dyes or radiotracers. The high concentration of “endogenous” water make it possible for wNMR to be contact-free in situ.

The essence of wNMR is a consistency check, which makes it useful for drug product manufacturing and inspection, where consistency is both critical and expected. For example, while wNMR cannot determine alum concentration directly, it can monitor consistency in alum filling and resuspension in vials. The same principle of consistency checking applies to monitoring emulsion droplet size, emulsion stability, antigen-alum complex stability, etc.

As defined herein, a “particle” corresponds to particles between about 1 nanometer and 50 microns in diameter, including particles that would be traditionally characterized as nanoparticles (e.g., about 1 nm to about 100 nm) and particles that would be traditionally characterized as micron-sized (e.g., up to about 10 microns). Particle size ranges relevant to the present invention include about 500 nm to about 1000 nm, about 1000 nm to about 5000 nm (5 microns), and about 1000 nm to about 10000 nm (10 microns). A person with an ordinary skill in the art will readily understand that the above particle size range is intended to be unlimited and could be extended to smaller and/or larger sizes. Also, it should be appreciated that the particles can be any shape, including spherical, can be substantially symmetrical or asymmetrical, and/or can be a single particle or be present as an aggregate of particles having an average aggregate size in a range from about 100 nm to about 10 microns or about 500 nm to about 1000 nm, about 1000 nm to about 5000 nm (5 microns), or about 1000 nm to about 10000 nm (10 microns). It should be understood by the person skilled in the art that the “particles” can be free aluminum particles or aluminum-antigen complexes, or a mixture of free aluminum particles and aluminum-antigen complexes.

As defined herein, the “alum-containing product” includes a product with nano- and micron-sized particles comprising aluminum and suspended in a solvent or a mixture of solvents. The alum-containing product can further comprise at least one surfactant, at least one water-soluble organic solvent, at least one dispersant, at least one biocide, at least one buffering agent, at least one pH adjusting agent (e.g., acids and/or bases), with or without antigens, or any combination thereof, as readily determined by the person skilled in the art. Many vaccines are alum-containing products because of the presence of an aluminum adjuvant.

As defined herein, a “vial” corresponds to a container, vessel, bottle, syringe, injection pen, or ampoule used to store the vaccine or other alum-containing product, wherein the vial comprises glass, plastic, ceramic, rubber, elastomeric material, and/or anything non-magnetic metal. The vial can have a screw top, a top that is closed using a cork or plastic stopper, a crimp vial (closed with a rubber stopper and a metal cap), a flip-top, a snap cap, or any other article of manufacture used to seal or close a vial. The vial can be tubular or cylindrical, or have a bottle-like shape with a neck. Other types and shapes of vials used to store particles as well as caps are readily understood by the person skilled in the art. The vials can be optically transparent or not optically transparent. There is no need to peel off any label on the vial, regardless of whether the label is transparent or not.

It is understood by the person skilled in the art that the “measuring” of the transverse relaxation rate of solvent R₂ is may be done by measuring some other parameter and converting to the R₂ value.

As defined herein, a “non-destructive” measurement is defined as a measurement performed without opening the vial or otherwise accessing the contents of the vial (for example by withdrawing a portion through a rubber gasket). Moreover, a non-destructive measurement means that no additives or probes or the like are added to the vial prior to the measurement of the transverse relaxation rate of solvent R₂ in the alum-containing product. Non-destructive also means that there is no need to make the vials optically transparent and no need to peel off any labels on the vials.

As defined herein, “alum” corresponds to aluminum-containing salts comprising one or more of aluminum hydroxide, aluminum phosphate, alum (KAl(SO₄)·12H₂O), aluminum hydroxyphosphate sulfate, as well as other known or proprietary aluminum salts that can be used as alum adjuvants or in pharmaceutical products comprising aluminum.

As defined herein, “DS” is a drug substance, which refers to the bulk drug solution or suspension or emulsion.

As defined herein, “DP” is a drug product, which is the combination of the DS plus the container or vial. In other words, DP refers to filled (partially or fully), sealed and labeled vials comprising the DS.

Suspensions and emulsions have complex hydrodynamic behavior, which might complicate the very last step of product manufacturing, the fill-finish step. For example, alum particles tend to sediment in water, which may lead to uneven filling of vials from the batch. As defined herein, the “evenness of alum particle filling levels” corresponds to a consistent alum particle concentration in each of the vials filled. For example, if the concentration of alum particles in the vial is expected to be a mean value of x mg/mL, then the evenness of alum particle filling levels corresponds to the assessment that the vial contains a mean value of x mg/mL±5% of the mean, which is typical of accepted United States Pharmacopeia (USP) variability If the alum particle concentration isn't consistent in all of the vials filled, pursuant to the specification of the alum-containing product, then product with too much or too little alum will be administered to the patient, with possible deleterious effects. More precisely, too much alum may cause harm while too little may render the alum-containing product, e.g., the vaccine, ineffective. Accordingly, if the alum particle concentration isn't consistent, i.e., the alum particle filling levels are not even, then the vial should not pass the quality control specifications. Notably, the method of using the transverse nuclear spin relaxation rate constant, R₂, of water, as described herein, cannot determine alum concentration directly, but instead monitors the consistency in the alum concentration from one vial to another.

The “extent of alum particle sedimentation” is a measure of whether the alum-containing product in the vial is properly resuspended following settling and/or transport and/or storage. Transport and storage may involve exposure of the alum-containing product to a variety of stressors including, but not limited to, temperature fluctuations, pH changes, vigorous shaking during transportation, exposure to sunlight, and freezing. The method of using the transverse nuclear spin relaxation rate constant, R₂, of water, as described herein, cannot quantitate the sedimentation directly, but instead monitors the consistency in the sedimentation over time and subsequent to possible stressors.

The present inventors have surprisingly discovered that solvent NMR can be used to monitor and detect the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in alum-containing products, e.g., vaccine products, in filled and sealed vials. Both the manufacturer as well as commercial end users and researchers can use solvent NMR to noninvasively inspect alum-containing products. In addition to being noninvasive, additional advantages of low field solvent NMR includes low cost instrumentation (e.g., a desktop NMR), simple and rapid data acquisition and analysis, minimal technical expertise, and results that are readily available within <5 min. It should be appreciated that the measurements can occur destructively as well, whereby the vial is opened, as readily determined by the person skilled in the art. Further, the method described herein can utilize high field NMR, if needed.

The method described herein is fast and reliable and allows the user to determine the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in alum-containing products, e.g., vaccines, as described herein.

In practice, the standard for evenness of alum particle filling levels and/or extent of alum particle sedimentation in the alum-containing product should be determined by the manufacturer. The manufacturer can provide the acceptable R₂(¹H₂O) range in sec⁻¹, i.e., a reference range, for the alum-containing product at a given temperature (e.g., 25° C.) and magnetic field strength (e.g., 0.5 T). The user will then measure the R₂(¹H₂O) of the alum-containing product at the same temperature and magnetic field strength and compare the measured R₂(¹H₂O) value with the manufacturer-specified acceptable range of R₂(¹H₂O), i.e., reference, as understood by the person skilled in the art, to determine if the alum particle filling levels were even (during the fill-finish step) and/or the alum-containing product has been resuspended properly (following storage and/or transport).

Accordingly, in a first aspect, a method of determining the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in an alum-containing product is described, said method comprising: measuring the transverse relaxation rate of solvent R_(2,m) in the alum-containing product; and determining the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in the alum-containing product by comparing the measured R_(2,m) to a reference transverse relaxation rate of solvent R_(2,r), wherein the reference R_(2,r) represents an acceptable range of evenly distributed aluminum particles or suspected aluminum particles, wherein when the measured R_(2,m) is inside the reference R_(2,r) range, the alum particle filling levels and/or the alum particle resuspension is acceptable. In one embodiment, the alum-containing product is a vaccine that comprises an alum-containing adjuvant. The transverse relaxation rate of solvent R₂ can be determined using solvent NMR, preferably low field solvent NMR. The measuring of the transverse relaxation rate of solvent R₂ in the alum-containing product can be done non-invasively in a vial. The reference R_(2,r) range, at a specified temperature, can be measured by the manufacturer and the result listed in the package insert and/or the vial of the alum-containing product. Preferably R_(2,m) is measured at substantially the same temperature as R_(2,r). The distributor or purchaser can then use NMR, e.g., benchtop, portable, or handheld, to measure R_(2,m) at the specified temperature and compare it with the reference R_(2,r) range listed in the package insert or vial before distribution or usage. If the evenness of alum particle filling levels and/or the extent of alum particle suspension is outside of the acceptable range determined by the manufacturer, and as such is unintended, that specific vial of alum-containing product should not be distributed or used immediately. It should be appreciated that if the evenness of alum particle filling levels and/or the extent of alum particle suspension is outside of the acceptable range determined by the manufacturer, the vial(s) can be shaken for additional time and the R_(2,m) remeasured to verify that the alum-containing product should be removed from distribution or use.

It should be appreciated that the transverse relaxation time T₂, which is the inverse of the transverse relaxation rate R₂, can be reported as the reference instead and the user uses the transverse relaxation time of solvent T₂ of the alum-containing product to determine the evenness of alum particle filling levels and/or the extent of alum particle sedimentation, as readily understood by the person skilled in the art. For example, in one embodiment of the first aspect, a method of determining the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in an alum-containing product using the transverse relaxation time T₂, of a solvent is described, said method comprising measuring the transverse relaxation time of solvent T_(2,m) in the alum-containing product, and determining the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in the alum-containing product by comparing the measured T_(2,m) to a reference transverse relaxation time of solvent T_(2,r), wherein the reference T_(2,r) represents an acceptable range of evenly distributed aluminum particles or suspected aluminum particles, wherein when the measured T_(2,m) is inside the reference T_(2,r) range, the evenness of the alum particle filling levels and/or the alum particle resuspension is acceptable. The alum-containing product can be a vaccine that comprises an alum-containing adjuvant. The transverse relaxation time of solvent T₂ can be determined using solvent NMR, preferably low field solvent NMR. The measuring of the transverse relaxation time of solvent T₂ in the alum-containing product can be done non-invasively in a vial. The reference T_(2,r) range, at a specified temperature, can be measured by the manufacturer and the result listed in the package insert and/or the vial of the alum-containing product. Preferably T_(2,m) is measured at substantially the same temperature as T_(2,r). The distributor or purchaser can then use NMR, e.g., benchtop, portable, or handheld, to measure T_(2,m) at the specified temperature and compare it with the reference T_(2,r) range listed in the package insert or vial before distribution or usage. If the evenness of alum particle filling levels and/or the extent of alum particle suspension is outside of the acceptable range determined by the manufacturer, and as such is unintended, that specific vial of alum-containing product should not be distributed or used immediately. It should be appreciated that if the evenness of alum particle filling levels and/or the extent of alum particle suspension is outside of the acceptable range determined by the manufacturer, the vial(s) can be shaken for additional time and the T_(2,m) remeasured to verify that the alum-containing product should be removed from distribution or use.

Although not wishing to be bound by this method, in one embodiment, i. the evenness is determined by verifying the alum level in the DS batch using destructive methods known in the art. Then the vials can be filled to obtain a DP. ii. Thereafter, the manufacturer can measure the R₂(¹H₂O) of some or all vials in the batch to determine if the vials have the same level of filling, e.g., are even or consistent. iii. Then, the DP level of a few vials in the batch can be verified using destructive methods to demonstrate that not only do all vials have the same filling level, but also the correct filling level. It should be appreciated that the order of steps ii and iii can be switched.

An example of the advantages associated with the methods described herein are as follows. The maximum allowed aluminum dose is 0.85 mg Al³⁺, according to the US Code of Federal Regulations [Vecchi, 2011]. The highest concentration of Al³⁺ in FDA-approved vaccines on the market is 1.2 mg/mL in BIOTHRAX. BIOTHRAX is packaged as 5-mL multi dose vials, wherein each dose is 0.6 mg/mL, below the federal limit of 0.85 mg/mL. The alum adjuvant in BIOTHRAX is aluminum hydroxide. If the vials are filled unevenly, and the deviation is +42%, then 0.5 mL will contain 0.852 mg or more Al³⁺, which exceeds the federal limit Advantageously, a 42% (or more) deviation is easily detected using the methods described herein. Further, if the multi-dose vial is resuspended improperly, then some doses may contain Al³⁺ higher than 0.6 mg and some doses may contain less, with the exact amounts unknown. Advantageously, the methods described herein can be used to detect alum resuspension levels before and after each dose is drawn from the vial, which allows the user to more accurately know the dose for each draw.

The present inventors have thus disclosed a nondestructive quality control technology using solvent NMR to determine the evenness of alum particle filling levels and/or the extent of alum particle sedimentation in alum-containing products, e.g., vaccines. This allows for the manufacturer and/or distributor, and/or end user to monitor for unexpected anomalies, e.g., too much or too little alum adjuvant, batch-to-batch variations of the same product, and/or if the alum-containing product was properly resuspended following transport and/or storage stressors. This can be done without opening the vial or peeling off the label. Further advantages include the applicability of the method for both continuous and batch unit manufacturing operations, both anti-bacterial and anti-viral vaccines, and/or both drug products and drug substances.

It should be appreciated that the methods defined herein are identifying a characteristic of the solutions that is different than the level of clustering or the extent of aggregation previously disclosed by the present inventors. “Aggregation” of biopharmaceuticals is understood to be driven by hydrophobic and/or hydrophilic interactions of proteins while “clustering” of particles is understood to be a phase separation of the particles from the solvent as a result of particle assembly which, unlike aggregation, is not driven by hydrophobic and/or hydrophilic interactions. In clustering, solvent molecules are trapped in the restricted compartments inside the clusters of particles. The currently described method involves sedimentation, which relates to the settling of more dense particles in a solvent, wherein if the particles are not evenly distributed in the solution, there is an uneven dose of the alum-containing product per unit volume. There is no known aggregation or clustering that impacts the evenness of filling or the resuspension of particles in the alum-containing product.

The features and advantages of the invention are more fully shown by the illustrative examples discussed below.

Example 1

The transverse relaxation rate of the water proton NMR signal, R₂(¹H₂O), response to the filling level of alum in sealed vials has been studied. Different concentrations of alum in water were used to simulate the variations of the alum filling levels under manufacturing conditions.

Five aqueous suspensions of alum in water with various alum concentrations were prepared at the Infectious Disease Research Institute (IDRI). The concentrations of alum in water were 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL, 2.0 mg/mL, and 5.0 mg/mL. Four mL of each alum suspension was introduced inside in a glass vial (4 mL max volume), sealed with rubber stopper, and secured by a metal cap.

The R₂(¹H₂O) in sec⁻¹ of each alum suspension was measured at 0.56 T (23.8 MHz ¹H resonance frequency) at 22° C. To measure the transverse relaxation rate constant R₂, Carr-Purcell-Meiboom-Gill (CPMG) experiments were used. It should be appreciated by the person skilled in the art that there are other methods to determine R₂ and the use of CPMG in the examples described herein are not intended to limit the determination of R₂. The transverse relaxation time T₂ (=1/R₂) value can be extracted by fitting experimental data to Formula (1):

I(t)=I ₀×exp(−t/T ₂)  (1)

where I(t) is the ¹H₂O signal intensity at time t, I₀ is the initial ¹H₂O signal intensity when t=0, and t is the T₂ delay time.

Measurements of R₂(¹H₂O) in sec⁻¹ were performed noninvasively, without opening the vial, since the wide bore (26 mm ID) of the probe of the low-field NMR spectrometer (0.56 T) permitted the accommodation of the vial without drawing a portion of the sample and transferring it into standard NMR tube.

The values of the transverse relaxation rate of water R₂(¹H₂O) measured in alum suspensions with different concentrations of alum are shown in FIG. 1 . FIG. 1 presents the results at 0.56 T from the CPMG pulse sequence at interpulse delay τ=300 μsec, where error bars represent the standard deviation of three consecutive measurements.

As seen from FIG. 1 , the values of the transverse relaxation rate of water R₂(¹H₂O) demonstrate strong sensitivity towards the changes in alum concentration in aqueous solutions. The slope the linear fit of the R₂(¹H₂O) vs. the alum concentration response, called relaxivity, is 1.07 (mg/mL)⁻¹·s⁻¹ for Alhydrogel® and 1.71 (mg/mL)⁻¹·s⁻¹ for Adju-Phos® at 0.56 T. To put matter in perspective, the relaxivity of nine FDA approved gadolinium-based contrast agents (GBCAs) is in the range of 4.26-8.54 (mg/mL)⁻¹·s⁻¹ at 0.47 T [Rohrer, 2005]. Considering alum salts are diamagnetic while GBCAs are paramagnetic, the particulate nature (micron-sized) of alum salts render them very efficient in causing water proton transverse relaxation.

Typically, the alum level in vaccine products is 0.5-1.0 mg/mL. A 5% filling deviation from 0.5 mg/mL is 0.025 mg/mL, which leads to a change of R₂(¹H₂O) of 0.027 s⁻¹. The error in R₂(¹H₂O) measurements is −0.0005 s⁻¹. This makes it possible to detect even <5% deviations in alum filling levels for the alum concentrations used in vaccine manufacturing.

Example 2

The transverse relaxation rate of the water proton NMR signal, R₂(¹H₂O), response to the sedimentation of the alum in sealed vials has been studied. Alum samples prepared and studied in the Example 1 with different concentrations of alum in water were used to study the sedimentation effects.

Five alum samples from Example 1 were kept undisturbed for 1 week at 4° C. to allow complete settling of the alum particles. After equilibration to 22° C., the sealed glass vials containing settled alum particles were carefully transferred into the probe of the NMR instrument for R₂(¹H₂O) detection. FIGS. 2(A) and 2(B) shows photos of a vial containing fully suspended alum particles and another vial containing fully settled alum particles, respectively.

The R₂(¹H₂O) of each alum suspension was measured at 0.56 T (23.8 MHz 1H resonance frequency) using Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. Data collection and processing were the same as described in the Example 1. Similar to the Example 1, the measurements of R₂(¹H₂O) were performed noninvasively, without opening the vial. It should be appreciated by the person skilled in the art that there are other methods to determine R₂ and the use of CPMG in the examples described herein are not intended to limit the determination of R₂.

The values of the transverse relaxation rate of water R₂(¹H₂O) measured in the settled alum particles with different concentrations of alum are shown in FIG. 3 . FIG. 3 compares the results of the R₂(¹H₂O) concentration dependence (circles) of fully suspended alum samples (i.e., Example 1) with the values R₂(¹H₂O) observed in fully sedimented alum samples (squares) at different concentrations. The error bars in FIG. 3 represent the standard deviation of three consecutive measurements and varies within 0.005-0.001 s⁻¹.

As seen from FIG. 3 , the values of the transverse relaxation rate of water R₂(¹H₂O) for settled alum particles are strikingly different from their fully suspended counterparts. This makes R₂(¹H₂O) a reliable and sensitive probe of the suspension uniformity in vaccine formulations.

Although the invention has been variously disclosed herein with reference to illustrative embodiments and features, it will be appreciated that the embodiments and features described hereinabove are not intended to limit the invention, and that other variations, modifications and other embodiments will suggest themselves to those of ordinary skill in the art, based on the disclosure herein. The invention therefore is to be broadly construed, as encompassing all such variations, modifications and alternative embodiments within the spirit and scope of the claims hereafter set forth.

REFERENCES

-   Baylor, N. W., Egan, W., Richman, P., (2002) Aluminum salts in     vaccines— US perspective. Vaccine, 20, S18-S23. -   Briggs, K. T., Taraban, M. B., Yu, Y. B. (2018) Water proton NMR     detection of amide hydrolysis and diglycine dimerization. Chem.     Comm. 54, 7003-7006. -   Feng, Y., Taraban, M. B., Yu, Y. B. (2011) Linear dependency of NMR     relaxation rates on shear modulus in hydrogels. Soft Matter, 7,     9890-9893. -   Feng, Y., Taraban, M. B., Yu, Y. B., (2015) Water Proton NMR—A     Sensitive Probe for Solute Association, Chem. Commun., 51,     6804-6807. -   Guo, J., Lewis, L. M., Billones, H., Tones, E., Kolhe, P., (2016)     The Effect of Shipping Stresses on Vaccine Re-dispersion Time.     Journal of Pharmaceutical Sciences, 105, 2009-2013. -   Kartoglu, U., Kenan Ozguler, N., Wolfson, L., Kurzatkowski,     W., (2010) Validation of the shake test for detecting freeze damage     to adsorbed vaccine. Bull. World Health Organ. 88, 624-631. -   Khatun, R., Hunter, H. N., Sheng, Y., Carpick, B W., Kirkitadze, M.     D., (2018) ²⁷Al and ³¹P NMR spectroscopy method development to     quantify aluminum phosphate in adjuvanted vaccine formulations.     Journal of Pharmaceutical and Biomedical Analysis, 159, 166-172. -   Levi, M. et al., (2017) The “Fluad Case” in Italy: Could it have     been dealt differently? Human Vaccines & Immunotherapeutics, 13,     379-384 -   Lewis, L. M., Guo, J., Tones, E., Wang, J., Billones, H., Kolhe, P.,     Young, A. L., Bates, D., Parker, A., Rigby-Singleton, S., (2017) Ex     Situ and In Situ Characterization of Vaccine Suspensions in     Pre-Filled Syringes. Journal of Pharmaceutical Sciences, 106,     2163-2167. -   Lindblad, E. B., (2004) Aluminum compounds for use in vaccines.     Immunology and Cell Biology, 82. 497-505, 82. -   Metz, H., Mäder, K., (2008) Benchtop-NMR and MRI—a new analytical     tool in drug delivery research, Int. J. Pharm., 364, 170-178. -   Mishra, A., Bhalla, S. R., Rawat, S., Bansal, V., Sehgal, R., Kumar,     S., (2007) Standardization and validation of a new atomic absorption     spectroscopy technique for determination and quantitation of     Aluminium adjuvant in immunobiologicals. Biologicals, 35, 277-284. -   Muthurania, K., Ignatius, A. A., Jin, Z., Williams, J., Ohtake,     S., (2015) Investigation of the sedimentation behavior of aluminum     phosphate: influence of pH, ionic strength, and model Antigens.     Journal of Pharmaceutical Sciences, 104, 3770-3781. -   Petrovsky, N. (2015) Comparative safety of vaccine adjuvants: a     summary of current evidence and future needs. Drug Safety, 38,     1059-1074. -   Rohrer, M, Bauer, H., Mintorovitch, Jan., Requardt, M., Weinmann, H.     J., (2005) Comparison of magnetic properties of MRI contrast media     solutions at different magnetic field strengths. Invest. Radiol. 40,     715-724. -   Signorelli, C., Odone, A., (2016) Dramatic 2015 excess mortality     rate in Italy: a 9.1% increase that needs to be explained. Scand. J.     Public Health, 44, 549-550. -   Taraban, M. B., Truong, H. C., Feng, Y., Jouravleva, E. V.,     Anisimov, M. A., Yu, Y. B., (2015) Water Proton NMR for In Situ     Detection of Insulin Aggregates, J. Pharm. Sci., 104, 4132-4141. -   Taraban, M. B., DePaz, R. A., Lobo, B., Yu, Y. B. (2017a) Water     proton NMR: a tool for protein aggregation characterization. Anal.     Chem. 89, 5494-5502. -   Taraban, M. B., Truong, H., Ilaysky, J., DePaz, R. A., Lobo, B.,     Yu, Y. B. (2017b) Non-invasive detection of nanoparticle clustering     by water proton NMR. Transl. Mater. Res. 4, 025002. -   Vecchi, S., Bufali, S., Skibinshi, D. A. G., O'hagan, D. T.,     Singh, M. (2011) Aluminum adjuvant dose guidelines in vaccine     formulation for preclinical evaluations. J. Pharm. Sci. 101, 17-20. -   Weerasekare, G. M., Taraban, M. B., Shi, X., Jeong, E.-K.,     Trewhella, J., Yu, Y. B. (2011) Sol and gel states in peptide     hydrogels visualized by Gd(III)-enhanced MRI. Biopolymers (Pept.     Sci.) 96, 734-743. -   Yu, Y. B., Feng, Y., Taraban, M. B. (2017) Water proton NMR for     noninvasive chemical analysis and drug product inspection. Am.     Pharmaceut. Rev. 20, 34-39. 

What is claimed is:
 1. A method of determining if an alum-containing product was acceptably resuspended following transport and/or storage, said method comprising: measuring a transverse relaxation rate of solvent R_(2,m) in the alum-containing product following transport and/or storage; and determining if the alum-containing product was properly resuspended by comparing the measured R_(2,m) to a reference transverse relaxation rate of solvent R_(2,r), wherein the reference R_(2,r) represents an acceptable range of evenly distributed aluminum particles or suspended aluminum particles, wherein when the measured R_(2,m) is inside the reference R_(2,r) range, the alum-containing product was acceptably resuspended.
 2. The method of claim 1, wherein R_(2,m) is measured using nuclear magnetic resonance (NMR).
 3. The method of claim 1, wherein the alum-containing product is contained in a vial, and wherein no additives or probes are added to the vial prior to measurement of the transverse relaxation rate of solvent R_(2,m).
 4. The method of claim 3, wherein R_(2,m) is measured without opening the vial or withdrawing the contents of the vial containing the alum-containing product.
 5. The method of claim 1, wherein the solvent is water.
 6. The method of claim 1, wherein the alum-containing product comprises particles in a range from 1 nanometer and 50 microns in diameter.
 7. The method of claim 1, wherein the alum-containing product comprises aluminum-containing salts selected from the group consisting of aluminum hydroxide, aluminum phosphate, alum (KAl(SO₄)·12H₂O), aluminum hydroxyphosphate sulfate, and any combination thereof.
 8. The method of claim 1, wherein the alum-containing product is a vaccine comprising alum adjuvants or a pharmaceutical product comprising aluminum particles.
 9. The method of claim 1, wherein acceptable resuspension indicates that the alum-containing product is useable or distributable as intended.
 10. The method of claim 1, wherein the alum-containing product comprises particles in a range from 1000 nm to 10000 nm.
 11. The method of claim 1, wherein R_(2,m) and R_(2,r) are measured at a substantially similar temperature.
 12. A method of determining if an alum-containing product was acceptably resuspended following transport and/or storage, said method comprising: measuring a transverse relaxation time of solvent T_(2,m) in the alum-containing product following transport and/or storage; and determining if the alum-containing product was properly resuspended by comparing the measured T_(2,m) to a reference transverse relaxation time of solvent T_(2,r), wherein the reference T_(2,r) represents an acceptable range of evenly distributed aluminum particles or suspended aluminum particles, wherein when the measured T_(2,m) is inside the reference T_(2,r) range, the alum-containing product was acceptably resuspended.
 13. The method of claim 12, wherein T_(2,m) is measured using nuclear magnetic resonance (NMR).
 14. The method of claim 13, wherein the NMR is portable.
 15. The method of claim 12, wherein the alum-containing product is contained in a vial, and wherein no additives or probes are added to the vial prior to measurement of the transverse relaxation time of solvent T_(2,m).
 16. The method of claim 15, wherein T_(2,m) is measured without opening the vial or withdrawing the contents of the vial containing the alum-containing product.
 17. The method of claim 12, wherein the solvent is water.
 18. The method of claim 12, wherein the alum-containing product comprises aluminum-containing salts selected from the group consisting of aluminum hydroxide, aluminum phosphate, alum (KAl(SO₄)·12H₂O), aluminum hydroxyphosphate sulfate, and any combination thereof.
 19. The method of claim 12, wherein the alum-containing product is a vaccine comprising alum adjuvants or a pharmaceutical product comprising aluminum particles.
 20. The method of claim 12, wherein acceptable resuspension indicates that the alum-containing product is useable or distributable as intended. 